The long range goal of this project is to understand the molecular mechanisms that control cellular differentiation during mammalian embryogenesis. Multipotent embryonal carcinoma cells are ideally suited for this purpose. These cells not only differentiate into a wide variety of cell types that are derived from the three embryonic germ layers, they also mimic, both morphologically and biochemically, important stages of early mammalian development. However, to fully utilize this model system one must be able to direct the differentiation of multipotent embryonal carcinoma cells to only one cell type. This project describes culture conditions under which two multipotent embryonal carcinoma cell lines differentiate primarily, and possibly exclusively, into parietal extraembryonic endoderm - one of the first cell types to appear during mammalian development. To avoid the inherent problems of serum-containing media, defined media have been developed that support the growth and/or the differentiation of several different embryonal carcinoma cell lines. The defined medium contains fibronectin, insulin, transferrin, and high density lipoprotein in place of serum. In this defined medium, multipotent embryonal carcinoma cells rapidly differentiate, with over 90% undergoing commitment within one cell cycle. After 72 hours, a pure population of differentiated cells has formed. More importantly, at least 85% of the cells exhibit the properties of parietal extraembryonic endoderm. Upon further examination of this new cell culture model, it was discovered that fibronectin and laminin promote the differentiation of multipotent embryonal carcinoma cells. This provided the first evidence that these molecules, which are developmentally regulated, may directly influence early mammalian development. This possibility is supported by the finding that early mouse embryos cultured in the absence of serum require fibronectin or laminin in order to develop beyond the blastocyst stage. Studies are in progress to determine the mechanisms by which fibronectin and laminin influence differentiation.